Individuals who suffer from the genetic disease xeroderma pigmentosum (XP) lack nucleotide excision repair (NER) of DNA, and thus have much higher carcinogenic probability than the average population after exposure to UV irradiation. NER is a major cellular biological defense system to remove DNA damage due to the formation of bulky lesions induced by UV irradiation and environmental genotoxic chemicals and carcinogens. Although, it has been generally accepted that DNA damage recognition plays a central role in NER, the molecular and thermodynamic details of DNA damage processing remain largely unclear, and have not been systematically studied using biochemically rigorous approaches. In addition, the roles of the damage recognition proteins XPA, RPA, and XPC-HR23B have been controversial. The long-term objective of this study is to understand the molecular and biochemical details of UV-induced or related DNA damage recognition and repair by human NER proteins, and the potential effects of these relationships on damage-induced mutagenesis and carcinogenesis. To gain a systematic and more precise view of DNA damage recognition and repair by human NER, the following questions will be addressed: What is the hierarchy of damage recognition and how is damage dynamically processed in a stepwise recognition mechanism? What structural and chemical alterations in the DNA helix are identified by repair proteins at specific recognition steps? What is the molecular architecture of recognition intermediates? And what protein domains are important for protein-DNA and protein-protein contacts in the recognition. Specifically, this project aims to determine the thermodynamics and kinetics of the interaction of XPC-HR23B, XPA, and RPA with DNA substrates containing site-specific UV photolesions and benzo[a]pyrene diol epoxide (BPDE) DNA adducts using rigorous biochemical approaches: identify and analyze the important protein motifs involved in damage recognition: characterize the repair intermediates for damage recognition of UV-induced photolesions and BPDE-DNA adducts; and determine the mechanism in which the structural and chemical modifications of damage are recognized and repaired.